US8879072B2 - Laser scanning microscope and method for operation thereof - Google Patents

Laser scanning microscope and method for operation thereof Download PDF

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Publication number
US8879072B2
US8879072B2 US13/413,960 US201213413960A US8879072B2 US 8879072 B2 US8879072 B2 US 8879072B2 US 201213413960 A US201213413960 A US 201213413960A US 8879072 B2 US8879072 B2 US 8879072B2
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intensity
channels
intensity values
detector
channel
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US20120229815A1 (en
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Nils Langholz
Dieter Huhse
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Carl Zeiss Microscopy GmbH
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Carl Zeiss Microscopy GmbH
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    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/008—Details of detection or image processing, including general computer control
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G02B21/0076—Optical details of the image generation arrangements using fluorescence or luminescence
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T5/00—Image enhancement or restoration
    • G06T5/50—Image enhancement or restoration using two or more images, e.g. averaging or subtraction
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T2207/00—Indexing scheme for image analysis or image enhancement
    • G06T2207/10—Image acquisition modality
    • G06T2207/10056—Microscopic image
    • G06T2207/10061—Microscopic image from scanning electron microscope
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T2207/00—Indexing scheme for image analysis or image enhancement
    • G06T2207/10—Image acquisition modality
    • G06T2207/10141—Special mode during image acquisition
    • G06T2207/10144—Varying exposure
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T2207/00—Indexing scheme for image analysis or image enhancement
    • G06T2207/20—Special algorithmic details
    • G06T2207/20172—Image enhancement details
    • G06T2207/20208—High dynamic range [HDR] image processing
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T2207/00—Indexing scheme for image analysis or image enhancement
    • G06T2207/20—Special algorithmic details
    • G06T2207/20212—Image combination
    • G06T2207/20216—Image averaging

Definitions

  • a fundus image with expanded dynamics is generated by means of a beamsplitter with an asymmetrical splitting ratio and a plurality of image sensors.
  • FIG. 1 schematically shows a beam path of a laser scanning microscope
  • FIG. 2 shows a an extracted view of a partial beam path in an embodiment of a detection arrangement
  • FIGS. 3 a )- 3 e ) show an X/Y brightness signal corresponding to a picture point along a direction Z resulting from a vertically proceeding recording of images;
  • FIG. 4 is a flowchart showing how the inventive advantageous evaluation of detector channels DE 1 , DE 2 in combination with the sample scanning is carried out by means of the laser scanning microscope;
  • FIG. 5 shows an embodiment using a graduated filter (VSD slide) provided in a laser scanning microscope
  • FIG. 6 shows the topography of a solar cell with pyramid structure recorded with a first channel
  • FIG. 7 shows the topography of a solar cell with pyramid structure recorded with a second channel
  • FIG. 8 sows the calculated topography of the two channels from FIGS. 6 and 7 .
  • a laser scanning microscope is basically made up of a plurality of modules: light source, scanning module, detection unit, and microscope. These modules are described in more detail in the following.
  • DE19702753A1 which is incorporated as reference in the disclosure.
  • lasers with different wavelengths are used in the light source module.
  • different lasers argon, argon krypton, TiSa lasers
  • the selection of wavelengths and the adjustment of the intensity of the required excitation wavelength are carried out in the light source module, e.g., using an acousto-optic crystal (AOTF).
  • AOTF acousto-optic crystal
  • the laser radiation generated in the light source is focused in the specimen in a diffraction-limited manner via the scanner, scanning optics and tube lens.
  • the focus scans the sample point by point in x-y direction.
  • the pixel dwell times during the scanning of the sample are usually in the range of less than one microsecond to several hundreds of microseconds.
  • the light which is emitted from the focus plane and from the planes situated above and below the latter arrive in a dichroic beamsplitter via the scanner.
  • This dichroic beamsplitter separates the sample light from the excitation light.
  • the sample light is then focused on a diaphragm (confocal diaphragm/pinhole) which is located precisely in a plane conjugate to the focus plane. In this way, light components outside the focus are suppressed.
  • a diaphragm confocal diaphragm/pinhole
  • Another dichroic block filter which further suppresses the illumination radiation is usually located behind the diaphragm.
  • the sample light is measured by a point detector (usually a photomultiplier tube (“PMT”)).
  • PMT photomultiplier tube
  • a number of different cell regions are labeled by different dyes simultaneously (multifluorescence).
  • the individual dyes can be detected separately based either on different absorption characteristics or emission characteristics (spectra).
  • an additional splitting of the fluorescent light of a plurality of dyes is carried out with the auxiliary beamsplitters (DBS), and a separate detection of the individual dye emissions is carried out in separate point detectors (e.g., PMTs).
  • DBS auxiliary beamsplitters
  • FIG. 2 is an extracted view of a partial beam path in the detection arrangement comprising pinhole optics with pinhole arranged therebetween and a partially transmitting beamsplitter ST for partial transmission in direction of a detector DE 1 , preferably a PMT, and partial reflection by a mirror SP in direction of a detector DE 2 , preferably a PMT.
  • the beam path in FIG. 2 can take the place of one or more detection beam paths in FIG. 1 .
  • the beamsplitter can be constructed as a 50:50 beamsplitter, but can preferably also have different splitting in directions DE 1 and DE 2 by means of corresponding coating, for example, 70:30, but also up to 99:1.
  • the beamsplitter ST is constructed so as to be displaceable (indicated by the arrow) relative to the beam path and has different splitting ratios along its path, for example, by means of different coatings; these splitting ratios can be formed discretely but also so as to pass into one another continuously so that, depending on the application, the splitting ratio can be changed continuously or discretely by displacing ST at an angle to the detection beam path.
  • either the sensitivity of the two detectors (PMT) is adjusted differently or the split beam is reduced in one of the beam paths to DE 1 and DE 2 , for example, by means of a reduction in transmission.
  • FIG. 4 shows how the inventive advantageous evaluation of detector channels DE 1 , DE 2 in combination with the sample scanning is carried out by means of the laser scanning microscope.
  • a point-by-point scanning of the sample by an illumination beam is generated by means of the LSM and the scanner thereof and the reflection signals or fluorescence signals corresponding to these illuminated points are acquired and associated with the respective picture point and stored as X values and Y values. Accordingly, an image is formed from a stored X/Y detection distribution. By moving the sample or the objective in (vertical) Z direction, these X/Y image distributions are recorded for different z values so that an X/Y/Z stack of images results after passing in Z direction and scanning in X/Y direction.
  • the recording is now carried out in a parallel manner with detectors DE 1 and DE 2 so that two separate image stacks which are associated with one another point by point are present in the image storage for these two detection channels.
  • the detection light is split to two different detectors with a sharply differing splitting ratio (e.g., 100 to 1).
  • a sharply differing splitting ratio e.g. 100 to 1.
  • the weak signal components can be made clearly visible in one channel, but the stronger signal components are overexposed or overdriven. However, these strong signal components are correctly measured in the second channel which is adjusted in such a way that there is no overexposure.
  • the two signals can then be combined and suitably calculated so as to obtain an image with higher dynamics or a topography image with fewer voids than each individual detector by itself would allow.
  • FIGS. 3 a - e An X/Y brightness signal corresponding to a picture point along a direction Z resulting from a vertically proceeding recording of images (see above) is shown in FIGS. 3 a - e by way of example for the two detection channels DE 1 , DE 2 .
  • a signal within the threshold value is registered by both detectors DE 1 and DE 2 .
  • the signal in DE 2 is so high that it lies above the upper threshold value; a signal is determined only in channel 1 (DE 1 ).
  • the signal falls below the lower threshold value in both DE 1 and DE 2 ; in 3 e ), the signal exceeds the upper threshold value in both channels.
  • FIG. 4 a shows the steps A 1 -A 3 common to both biomedical image recordings and topography recordings.
  • FIG. 4 b shows the sequence in A 4 -A 6 in topography measurement, and
  • FIG. 4 c shows steps A 5 , A 6 in biomedical imaging.
  • splitting either of light or sensitivities
  • greater total dynamics are obtained.
  • knowledge of the exact splitting factor or even a calibration of the two channels relative to one another is not necessary.
  • a graduated filter shown in FIG. 5 , which is provided in the LSM 700 can now also be used according to the invention.
  • this graduated filter (see also EP1882969 A1) has a glass plate which was used heretofore to effectively “switch off” the graduated filter. It has a fully reflecting mirror (mirror) at its other edge.
  • the glass plate can surprisingly be used as beamsplitter ST as described in FIGS. 2 , 3 by its splitting ratio of approximately 99:1.
  • the gain for the two channels should be selected in such a way that overexposures do not occur in the 1-% channel.
  • the gain of the 99-% channel should be selected in such a way that there are enough voxels which are neither overexposed nor underexposed in the two channels.
  • An overview image stack is preferably recorded initially and the occurring maximum and minimum intensity is determined.
  • the overview stack can also be carried out with lower resolution (fewer X/Y or Z points are displayed).
  • measuring is carried out with both channels simultaneously.
  • the calculation of the two individual channels to one channel takes place following the measurement.
  • biomedical evaluations and topography evaluations can be advantageously distinguished.
  • the two approaches are described in the following.
  • prior calibration of the measurement system is advantageous, particularly for the beamsplitter which is used and possibly for the wavelength dependency thereof or for the differently adjusted detector gain, and the measurement is then evaluated in a wavelength-dependent manner.
  • FIGS. 6-8 In topography measurements, the height evaluation is advantageously carried out separately for every x,y coordinate. Channel assignment should be oriented towards height evaluation. This is shown in FIGS. 6-8 .
  • FIG. 6 was recorded with channel 1 in FIG. 2 and with channel 2 in FIG. 7 .
  • the calculated image is shown in FIG. 8 .
  • FIG. 6 shows the topography of a solar cell with pyramid structure.
  • An electrode of the solar cell can be seen in the upper right-hand area of the image. This electrode has a very high reflectivity in contrast to the solar cell matrix.
  • the first channel was adjusted based on an overview image in such a way that the brightest reflections on the electrode lie within the dynamic range of the PMT.
  • the white areas (particularly at lower left) show the places where there is too little (or too much) light for a useful evaluation.
  • the adjustment of the channel is carried out, for example, in that in addition to the (for example) 99:1 split between channel 1 and channel 2 , a change in gain, for example, for channel 1 , is carried out so that this channel has a sensitivity which is, for example, ten times higher than the other channel so that at a splitting ratio of 99:1 a ratio of about 1000:1 is adjusted between the two channels through the change in sensitivity in order to include as many picture points as possible in the applicable area between Su and So so that a picture point can be formed in the calculated image.
  • the topography of the solar cell is shown again in FIG. 7 .
  • the difference between FIG. 6 and FIG. 7 is that measurement was carried out with the second channel.
  • the channel splitting was carried out by the mirror position of the VSD slide.
  • the second channel was adjusted as was described above in such a way that there is another overlapping area at the applicable pixels for the subsequent calculation.
  • the overexposed or underexposed pixels are again identified by white in FIG. 7 .
  • the measurements in the two channels took place simultaneously.
  • the calculated topography of the two channels is shown in FIG. 8 .
  • the average of the two height values was calculated in the common applicable area of the two channels. When there was only one applicable height value, this was used.
  • the white positions in the image show the locations in which there is an overexposure or underexposure on both channels.
  • the example shows the case of a fast measurement and no scenario with optimized measuring conditions. Accordingly, further improvements are possible.

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  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Optics & Photonics (AREA)
  • Health & Medical Sciences (AREA)
  • Pathology (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Computer Vision & Pattern Recognition (AREA)
  • General Engineering & Computer Science (AREA)
  • Theoretical Computer Science (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Microscoopes, Condenser (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)
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US13/413,960 2011-03-08 2012-03-07 Laser scanning microscope and method for operation thereof Active 2032-10-02 US8879072B2 (en)

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DE102011013614A DE102011013614A1 (de) 2011-03-08 2011-03-08 Laser-Scanning-Mikroskop und Verfahren zu seinem Betrieb
DE102011013614.2 2011-03-08
DE102011013614 2011-03-08

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Cited By (4)

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WO2017105649A1 (fr) * 2015-12-17 2017-06-22 Bae Systems Information And Electronic Systems Integration Inc. Capteur imageur et système d'alignement laser
US10146039B2 (en) 2013-07-04 2018-12-04 Leica Microsystems (Schweiz) Ag Image capture method for a microscope system, and corresponding microscope system
US20190179127A1 (en) * 2017-12-12 2019-06-13 Trustees Of Boston University Multi-z confocal imaging system
US11379954B2 (en) * 2019-04-17 2022-07-05 Leica Instruments (Singapore) Pte. Ltd. Signal to noise ratio adjustment circuit, signal to noise ratio adjustment method and signal to noise ratio adjustment program

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JP6257156B2 (ja) * 2013-03-04 2018-01-10 オリンパス株式会社 顕微鏡装置
JP6289044B2 (ja) * 2013-11-15 2018-03-07 オリンパス株式会社 観察装置
LU92665B1 (de) * 2015-02-24 2016-08-25 Leica Microsystems Verfahren zur verbesserung des dynamikbereichs einer vorrichtung zum detektieren von licht
US10184835B2 (en) * 2015-09-23 2019-01-22 Agilent Technologies, Inc. High dynamic range infrared imaging spectroscopy
DE102016101832A1 (de) * 2016-02-02 2017-08-03 Frt Gmbh Verfahren und Messvorrichtung zur Messung der Topographie unter Verwendung von mindestens zwei Höhenebenen einer Oberfläche
TWI820406B (zh) * 2021-03-24 2023-11-01 立克銘白生醫股份有限公司 用於檢測生物粒子的檢測設備及檢測設備的檢測方法

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US10146039B2 (en) 2013-07-04 2018-12-04 Leica Microsystems (Schweiz) Ag Image capture method for a microscope system, and corresponding microscope system
WO2017105649A1 (fr) * 2015-12-17 2017-06-22 Bae Systems Information And Electronic Systems Integration Inc. Capteur imageur et système d'alignement laser
US20190003828A1 (en) * 2015-12-17 2019-01-03 Bae Systems Information And Electronic Systems Integration Inc. Sensor imager and laser alignment system
US10466044B2 (en) * 2015-12-17 2019-11-05 Bae Systems Information And Electronic Systems Integration Inc. Sensor imager and laser alignment system
US20190179127A1 (en) * 2017-12-12 2019-06-13 Trustees Of Boston University Multi-z confocal imaging system
US11042016B2 (en) * 2017-12-12 2021-06-22 Trustees Of Boston University Multi-Z confocal imaging system
US11379954B2 (en) * 2019-04-17 2022-07-05 Leica Instruments (Singapore) Pte. Ltd. Signal to noise ratio adjustment circuit, signal to noise ratio adjustment method and signal to noise ratio adjustment program

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JP2012190021A (ja) 2012-10-04
US20120229815A1 (en) 2012-09-13
EP2497412B1 (fr) 2016-10-05
DE102011013614A1 (de) 2012-09-13
EP2497412A1 (fr) 2012-09-12

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